PI3K/Akt Pathway Protects Cardiomyocytes From Apoptosis After Severe Burns

Our previous studies demonstrated that myocardial damage occurs immediately following severe burns even before significant reduction in blood volume.Such myocardial damage leads to cardiac deficiency,and it is also a precipitating factor for burn shock and ischemic/hypoxic injury.Therefore,we called it 'shock heart'.PI3K is one of the most important intracellular signal transductuction molecules.After PI3K is activated,the second messenger PIP3 is produced at plasma menmbranes,which phosphorylates Akt at Ser308.The activated Akt further phosphorylates the target proteins, such as Bad,Caspase9,NF-kB,GSK-3,FKHR,p21^Cip1 and p27^Kip to modulate the cell proliferation,differentiation,apoptosis and immigration.The expression,function and potential mechanism of PI3K/Akt pathway in myocardium at early postburn stage remains unlear.We postulated that PI3K/Akt pathway is activated at the same time when the cardiocytes are inflicted with hypoxic/ischemic injury at early stage following severe burns,which may have a anti-apoptotic effect by modulation of calcium,mitochondrial membrane potential,HIF-1αexpression and apoptotic gene expression.The present study was designed to confirm this hypothesis.Material and MethodsMethods:Both in vivo and in vitro experiments were adopted in the present study.The animal model was established by scald in rats.The PI3K mRNA expression of myocardium was detected by RT-PCR.PI3K and phosphorylated Akt protein expression were detected by western blot.To elucidate the function of PI3K/Akt signal pathway in cardiomyocytes during hypoxia and ischemia,the in vitro model was established by cultured rat cardiomyocytes maintaining in hypoxia and ischemia environment.With treatment of specific PI3K/Akt inhibitor LY294002 and selective PI3K/Akt activator IGF-1,ELISA,laser scanning confocal fluorescence microscope,TUNEL technique were used to observe the influence of PI3K/Akt pathway on cardiomyocyte viability,LDH activity,mitochondrial membrane potential,inner Ca²⁺ concentration and apoptotic rate.LY294002 and Rapamycin treatment were used to explore the possible mechanism of apoptosis.Alteration of the apoptotic gene expression such as P53,C-myc,Bax,Bcl-2 and HIF-1αmRNA were detected by RT-PCR and/or Western blot.Results and Conclusion:1.PI3K mRNA and protein expression and Akt phospharylation were upregulated significantly at early stage of severe burn,which showed the PI3K/Akt pathway was activated.2.Hypoxia and ischemia induced progresive decrease of cardiocyte viability.The decrease of cardiocyte viability was more severe after PI3K/Akt inhibitor LY294002 was used,indicating that the PI3K/Akt pathway plays a role in the maintainance of cell viability and protection of cells from damage induced by hypoxia and ischemia.3.Hypoxia and ischemia caused the membrance damage of cardiocytes refleted by increase of LDH leakage.After pretreatment with LY294002,the LDH was obviously increased.It suggests that PI3K/Akt pathway can maintain the cardiocyte membrane stability.4.The apoptotic rate in cardiocytes cultured under hypoxia and ischemia environment was increased significantly,and was intensified by LY294002 pre-treatment.When PI3K/Akt activator IGF-1 was given,the apoptotic rate was decreaed.This result suggests that the PI3K/Akt pathway can protect cardiocytes from apoptosis as an endogenous cytoprotective factor.5.At 6h after hypoxia and ischemia,inner Ca²⁺ concentration increased obviously, which was intensified by LY294002 pre-treatment.When PI3K/Akt activator IGF-1 was given,concentration of inner Ca²⁺ was decreased,indicating that the activation of PI3K/Akt can relieve the Ca²⁺ overload and the consequent apoptotic reaction.6.At 6h and 12h after hypoxia and ischemia,mitochondrial membrane potential decreased obviously,which was also intensified by LY294002 pre-treatment.When PI3K/Akt activator IGF-1 was given,the decreasing of mitochondrial membrane potential was alleviated,suggesting that PI3K/Akt pathway can maintain the stability of mitochondrial membrane and prevent the apoptotic reaction induced by MPTP openning of mitochondria.7.PI3K/Akt/mTOR/p70S6K pathway may play a role in regulating HIF-1αexpression in mRNA and protein level to prevent cardiocyte apoptosis under hypoxia and ischemia condition.8.The activation of PI3K/Akt,through upregulation of HIF-1αexpression,further downregulated the wildtype P53 expression in cardiocytes,raised the Bcl-2/Bax ratio, upregulated C-myc expression so as to protect cells from apoptosis under hypoxia and ischemia condition.